Transcription factors represent a group of molecules within the cell that mediate the connections between extracellular signals and intracellular responses culminating in altered gene expression. Immediately after an environmental stimulus, these proteins which reside predominantly in the cytosol are translocated to the nucleus where they bind to specific DNA sequences in the promoter elements of target genes and activate the transcription of these target genes. One family of transcription factors, E2F transcription factors, regulates the expression of an extensive panel of genes that control DNA synthesis and cellular proliferation in a cell cycle-dependent manner reviewed in (Dyson, Genes & Development, 1998, 12, 2245-2262; Lavia and Jansen-Durr, BioEssays, 1999, 21, 221-230; Yamasaki, Results Probl. Cell. Differ., 1998, 22, 199-227).
The deregulation of the expression or function of E2F proteins alone or in conjunction other E2F member proteins has been implicated in the development of certain pathologic conditions, including cancer (Sladek, Cell. Prolif., 1997, 30, 97-105). The pharmacological modulation of E2F protein activity is therefore believed to be an appropriate point of therapeutic intervention. Hayes et al. have recently discovered that the damage-specific DNA binding protein, DDB, can function as a transcriptional partner to E2F1 (Hayes et al., Mol. Cell. Biol., 1998, 18, 240-249) therefore making it a potential target for E2F regulation. In their studies they demonstrate that DDB associates with the activation domain of E2F1 in HeLa cell extracts and in living cells, that DDB stimulates E2F1-activated transcription and that DDB expression can overcome the retinoblastoma protein (Rb)-mediated inhibition of E2F1-activated transcription. Taken together, these data suggest that DDB might act as a negative regulator or E2F.
DDB (also known as UV-DDB, XAP-1 for Hepatitis B virus X-associated protein 1, XPE-BF for xeroderma pigmentosum group E UV-damaged DNA binding factor) is a heterodimeric protein composed of a 48 and 127 kD subunit which are also known as p48 and p127 as well as DDB1 and DDB2, respectively. Chromosomal mapping studies have shown that the two subunits are products of different genes, both located on human chromosome 11 (Dualan et al., Genomics, 1995, 29, 62-69).
DDB was first identified as a factor that binds UV-damaged DNA and whose activity is missing in cells from a subset of xeroderma pigmentosum complementation group E (XP-E) patients (Hwang et al., Mutat. Res., 1996, 362, 105-117; Keeney et al., J. Biol. Chem., 1993, 268, 21293-21300). This defect was shown to be corrected by injection of the DDB protein into cells from XP-E patients (Keeney et al., Proc. Natl. Acad. Sci. U.S. A., 1994, 91, 4053-4056).
DDB has been shown to interact with other cellular and viral factors including the cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (Watanabe et al., J. Neurochem., 1999, 72, 549-556), cullin 4A, a ubiquitin ligase upregulated in breast cancer (Shiyanov et al., J. Biol. Chem., 1999, 274, 35309-35312), the V protein of the paramyxovirus (Lin et al., Virology, 1998, 249, 189-200) and the X protein of the hepatitis B virus (Butel et al., Princess Takamatsu Symp., 1995, 25, 185-198).
The interaction of DDB with these proteins as well as with E2F1 have been shown to be mediated by the p48 subunit of DDB. Characterization of the subunits by several groups has shown that p48 expression is upregulated as early as 38 hours post UV irradiation (Nichols et al., J. Biol. Chem., 2000, 275, 21422-21428) and that when expressed independently the p48 subunit localizes to the nucleus while the p127 subunit localizes to the cytoplasm (Shiyanov et al., Mol. Cell. Biol., 1999, 19, 4935-4943). These studies also showed that p48 plays a critical role in the nuclear localization of p127. This role is compromised when p48 contains the mutations of the XPE group (Shiyanov et al., Mol. Cell. Biol., 1999, 19, 4935-4943) and DNA binding and repair is incomplete when a nonsense mutation occurs in the gene (Itoh et al., J. Invest. Dermatol., 1999, 113, 251-257).
The p48 subunit has also been linked to the regulation of the TGF-beta signaling in human lung cancer cells. DDB2 (p48) is one of several genes that is upregulated upon p53 induction (Kannan et al., FEBS Lett., 2000, 470, 77-82) and expression of p48 strongly depend on basal p53 expression (Hwang et al., Proc. Natl. Acad. Sci. U. S. A., 1999, 96, 424-428).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of DDB and to date, strategies aimed at investigating DDB function have involved the use of antibodies, modulation of p53 expression, and differential expression of the two subunits. Consequently, there remains a long felt need for agents capable of effectively and specifically inhibiting DDB1 and/or DDB2 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of DDB1 (p127) expression.
The present invention provides compositions and methods for modulating DDB1 (p127) expression.